THE GOAL of the proposed research is to develop a platform for DNA analysis, start the PI as a young investigator in health-related research, train students, and strengthen the research environment of the institution. Assuming a target DNA molecule has multiple binding sites to a particular fluorescent sensing probe, the relative locations of the binding sites on the DNA contain rich information about the identity of the target DNA. However, the traditional sensing platforms have difficulties to tell the relative locations of the binding sites on the DNA, either because the DNA is tangled into a random coil or there are technical challenges in getting high spatial resolution. Here I propose to combine the super-resolution light microscopy and genome optical mapping to resolve these challenges. The key idea is to stretch the target DNA on a substrate, use triplex-forming oligonucleotide (TFO) as probes and a super-resolution optical nanoscopy to observe weak but repetitive probe-target binding time trajectories of each specific location on the target DNA. More specific goals of the new platform are: (1) to achieve mapping resolution to single-digit nanometer (corresponding to a few tens of bases); (2) to achieve the tunability of mapping density to 10-1000 bases; (3) to identify false positive sites and false negative sites by evaluating the binding kinetics of the labeling probes; (4) to motivate and educate the graduate, undergraduate, and high school students in biophysical research. In order to achieve these goals, chemistry and surface chemistry problems have to be solved and special data analysis strategies have to be developed. As such, the following specific aims will be pursued: 1. Stretch and immobilize the single double-stranded DNA (dsDNA) molecules. The strategies are detailed in this proposal, with preliminary results confirming the stretched phage lambda DNAs. 2. Develop TFO probes and compare the new platform to the traditional methods. TFOs will be evaluated with the lambda DNA and further tested with the E. coli DNA. The optical maps will be compared to those obtained with the traditional method. Software tools have been developed by the PI to evaluate and optimize the probes, as well as to analyze the super-resolution data and to plot the optical maps. 3. Distinguish specific and false-positive binding sites. The probe-target interactions are captured at the single-molecule level in this new platform. The binding kinetics is resolved for each binding sites. Preliminary simulations of the dynamics have been performed and the software to analyze these data has been validated. This project will benefit genomic analysis of eukaryotes (e.g. human, animal, fish, plant, and fungi) and prokaryotes (e.g. super bacteria, HIV, Herpes, and Zika viruses). Several graduate and undergraduate students will be trained in this project. The PI has specific training plans. Parts of the project have been incorporated into an undergraduate laboratory course.